Collagen peptide modified carboxymethyl cellulose as both antioxidant drug and carrier for drug delivery against retinal ischaemia/reperfusion injury

Abstract Oxidative stress can cause injury in retinal endothelial cells. Carboxymethyl cellulose modified with collagen peptide (CMCC) is of a distinct antioxidant capacity and potentially a good drug carrier. In this study, the protective effects of CMCC against H2O2‐induced injury of primary retinal endothelial cells were investigated. In vitro, we demonstrated that CMCC significantly promoted viability of H2O2‐treated cells, efficiently restrained cellular reactive oxygen species (ROS) production and cell apoptosis. Then, the CMCC was employed as both drug and anti‐inflammatory drug carrier for treatment of retinal ischaemia/reperfusion (I/R) in rats. Animals were treated with CMCC or interleukin‐10‐loaded CMCC (IL‐10@CMCC), respectively. In comparisons, the IL‐10@CMCC treatment exhibited superior therapeutic effects, including better restoration of retinal structural thickness and less retinal apoptosis. Also, chemiluminescence demonstrated that transplantation of IL‐10@CMCC markedly reduced the retinal oxidative stress level compared with CMCC alone and potently recovered the activities of typical antioxidant enzymes, SOD and CAT. Therefore, it could be concluded that CMCC provides a promising platform to enhance the drug‐based therapy for I/R‐related retinal injury.

carboxymethyl cellulose, as functional medical materials, has been found to be able to reduce the high level of ROS. 14,15 However, this antioxidative capability of carboxymethyl cellulose against ROS was rather limited. 16 An improving approach was modifying the cellulose with a stronger antioxidative group. Collagen peptide was known for its abundant physiological functions, such as chemotaxis, platelet aggregation, suppressing osteoclast differentiation and, in particular, protecting against oxidative free radical. [17][18][19] Thus, the functionalization of carboxymethyl cellulose with collagen was expected to effectively improve its antioxidative capacity.
IL-10, a potent anti-inflammatory cytokine, is known for its potent capacity that hampered inflammation and modulated pathogenesis of inflammatory syndromes. 20 The IL-10 has been found to play a critical protective role in multiple inflammatory diseases, such as inflammatory bowel disease, experimental allergic encephalomyelitis and atherosclerosis. [21][22][23] In this study, we prepared carboxymethyl cellulose grafted collagen peptides and investigated its antioxidative property in vitro against H 2 O 2 -simulated oxidative stress environment. Then, IL-10loaded antioxidative gels were delivered into the eyes of rats after retinal I/R injury for practical attempts. In addition, we preliminarily studied the underlying mechanism of enhanced restoration of retina from apoptosis with IL-10-loaded antioxidative gels by investigating the level of antioxidant enzyme and the mRNA expression of inflammatory cytokines.

| Preparation of carboxymethyl cellulose grafted collagen peptide
The synthetic approach was based on the previous method. 24 Typically, first, carboxymethyl cellulose was first dissolved with magnetic stirring, followed by NHS-EDC coupling reaction (EDC:CMC, 1:2).
Then, after reacting for 1 hour, collagen peptide with the mass ratio of 5:3 to CMC was added into the mixture and the grafting reaction was undertaken in MES buffer (pH 6) for 20 hours at 55°C with a constant vigorous stirring. The final products, carboxymethyl cellulose grafted collagen peptide (CMCC), were lyophilized and stored before dialysis for 3 days.

| Isolation and culture, viability and identification of retina endothelial cells
Three-week-old male Sprague-Dawley (SD) rats were applied to the isolation of primary retina endothelial cells (rRECs), according to previous protocol. 25 Typically, rats eyes were first enucleated and hemisected. The retinas were separated with a dissecting microscope, immersed in HBSS in the presence of penicillin/streptomycin (Sigma) and rinsed. A total of twenty-four retinas were undertaken digestion with 0.5% collagenase type I (Worthington, NJ) in serum-free DMEM for 45 minutes at 37°C. Then, endothelial cell-specified medium containing 1% ECM (5% foetal bovine serum, 100 mg/mL streptomycin and 100 U/mL penicillin ScienCell Research Laboratories, CA) was supplemented. The obtained cells undertook filtering through 70and 40-μm nylon mesh (Becton, USA), successively, and were redispersed in ECM followed by incubating with goat antimouse magnetic beads pre-coated via mouse anti-rat PECAM-1. After thoroughly washing with HBSS for 6 times and resuspending in ECM, the beads contained solution was then seeded on 6-well plates pre-treated with human fibronectin (Thermo Fisher Scientific, USA).
The viability of the resultant cells underwent propidium iodide (PI) evaluation for viability by flow cytometry, according to previously reported method. 26 Briefly, after incubation with PI (100 ng/ mL) in phosphate-buffered saline (PBS) for 1 minute, cells were centrifuged to remove free PI and then resuspended in PBS containing 2% FBS for analysis using a BD LSR II flow cytometer (BD Biosciences, USA). Viable cells (PI cells) were calculated by flow cytometry at a constant flow rate. rRECs were confirmed by a positive immunofluorescence staining for endothelial cells markers: factor VIII-related antigen. Briefly, cells, grown in cell slides of the 24-well plates, were fixed with 4% paraformaldehyde for 10 minutes. After rinsing with PBS, they were blocked with 5% normal goat serum in PBS for 30 minutes and incubated with rabbit anti-factor VIII-related antigen polyclonal antibody (Santa Cruz, CA, USA) at 4°C overnight.
Cells then were incubated with rhodamine-conjugated anti-rabbit secondary antibody at 37°C for 30 minutes. Afterwards, cells were washed with PBS 3 times and were staining with 4'6-diamidino-2phenylindole (DAPI, Sigma) for 5 minutes. The resulting samples were examined using a Olympus FV1000 confocal laser scanning microscope. Eight replicates were prepared for each treatment group.

| Determination of intracellular reactive oxygen species
The intracellular ROSs were analysed using dihydroethidium (DHE) according to the previous protocol. Typically, rRECs were added with 1 mM DHE, incubating at 37°C for 25 minutes. The suspension was gently washed with PBS buffers for 3 times. The stained cells were then harvested and underwent FACScan flow cytometer (BD Biosciences) analysis.

| Terminal deoxynucleotidyl transferase UTP nick end labelling (TUNEL) assay
TUNEL assay kits were purchased from Beyotime Biotechnology, China, and were performed as described in the manufacturer's protocols. Typically, the air-dried rRECs were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 containing 0.1% sodium citrate. Then, these cells were darkly incubated with TUNEL reagent. After washing, the cells were casted onto glass slides and immersed with a DAPI-containing antifade mounting medium.

| Western blotting assay
The proteins of lysed rRECs and retina were analysed using BCA TM Protein Assay Kit (Pierce, USA), according to the manufacturer's protocol. Briefly, protein sample was first separated with SDS-PAGE approach. The discrete protein strips were then electrophoretically transferred to nitrocellulose membranes, followed by incubating with primary antibodies against cleaved caspase 3 (Csp3), Bax, Bcl-2, TNF-α, IL-1β, iNOS, ICAM-1 and MCP-1 overnight, respectively, and then the corresponding secondary antibodies marked with HRP for 1 hour. β-Action and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as internal reference for these cytokines. The corresponding antibodies were purchased from Cell Signal Technology, USA.

| IL-10 loading and releasing assay in vitro
The IL-10 loading CMCC (IL-10@CMCC) was obtained by soaking CMCC in saturated IL-10 solution overnight at 4°C. The releasing assay of IL-10@CMCC was performed using ELISA to detect the released IL-10. Briefly, aliquots of the IL-10@CMCC were separately dispersed and standing in 5 mL 1 × PBS (pH7.4). Subsequently, the supernatants were collected and analysed at different days. Each sample was repeated 3 times.

| Retinal ischaemia/reperfusion injury
Sprague-Dawley rats with similar weights were anesthetized using sodium pentobarbital and then received surgery, performed as described previously. 27 Briefly, the anterior chambers of rat eyes were cannulated utilizing a 27-gauge infusion needle connected with a normal saline reservoir. The intraocular pressure was then tuned up to 110 mm Hg for 1 hours. The retinal ischaemia/reperfusion was verified through examining the fundus whitening and the retinal blood flow restoration.

| Measurement of retinal thickness
The retinal thickness of rats was measured as previously described. 29 In brief, the rats were first anaesthetized at day 21, followed by the enu-

| Immunofluorescence staining of Csp3
Typically, the cut-off retinal sections received fixing with 4% paraformaldehyde for 10 minutes, permeabilizing with 0.1% Triton X-100 for 15 minutes and blocking with 5% normal goat serum for 60 minutes. rRECs were then incubated with Csp3 antibody (1/200 diluted) overnight and secondary antibody modified with Alexa Fluor 568 for 1 hour (1/500 diluted, Molecular Probes, USA). DAPI staining was used as reference. Images were also attained via the Flouview-FV300 Laser Scanning Confocal system.

| Inflammation level assessment via quantitative polymerase chain reaction (PCR)
The extraction of total RNA from the retina was performed using TRIzol reagent (Invitrogen-Life, USA). Then, 1 μg of total RNA was annealed with 300 ng of oligo (dT) (Promega, USA) for 5 minutes at 65°C, followed by reversely transcribing to cDNA utilizing 80U Moloney murine leukaemia virus reverse transcriptase (Gibco-life, USA) at 37°C for 1 hour. The obtained cDNA was applied to realtime qPCR using iTaq Universal SYBR Green Supermix (Bio-Rad) using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad).

| Measurements of the activities of antioxidant enzymes in retina
Colorimetric methods were aplicated to evaluate superoxide dismutase (SOD) and catalase (CAT) activities using kits (Cayman Chemical, USA) in the retina at day 3, 7 and 14. Assays were conducted according to the manufacturer's protocols.

| Statistical analysis
Digital data were expressed as mean ± SD. Statistical significance was denoted as P value < .05. The analysis within groups employed one-way ANOVAs followed by Tukey's post hoc test for multiple pairwise tests.

| rRECs viability and identification
As can be seen in Figure 1A, the morphology of the isolated rRECs was fusiform. The PI evaluation showed that viable cells (PI-) reached 98.8%, indicating desirable isolation efficiency ( Figure 1B). Furthermore, the obtained cells were immunofluorescence stained with the specific biomarker of rRECs (factor VIII) and DAPI for cell nucleus. As shown in Figure 1C, the almost complete overlapping of the 2 markers indicated the high purity of rRECs.

| CMCC protect rRECs against H 2 O 2 -induced oxidative stress
The introduction of H 2 O 2 into rRECs culture medium was used to establish in vitro oxidative stress model and assess whether CMCC was able to strengthen the viability of rRECs under the condition. As shown in Figure 1D The CMCC was known for its antioxidative capability. As predicted, the 500 μmol/L H 2 O 2 -induced decrease in rRECs viability can be considerably offset by the addition of this antioxidative gels ( Figure 1E). Furthermore, as shown in Figure 1F

| Assessment of apoptotic proteins
The investigation of apoptotic proteins expression in rRECs further validated the underlying mechanism. As shown in Figure 3B, H 2 O 2induced oxidative stress significantly up-regulated pro-apoptotic protein, Csp3 and the ratio of Bax to Bcl-2. This modulation in rRECs was then significantly suppressed when the antioxidative gels, CCPCC, were introduced.

| In vitro assay of IL-10 releasing
The released IL-10 in supernatant was measured at days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, respectively (Figure 4). It can be seen that MU ET AL. | 5011 about 25% amount of drugs were released at the first day. With time, the releasing speed was correspondingly decreased. At day 10, only 2% increase was detected, compared with the releasing amount at day 9, indicating the relative retention of loaded IL-10 in antioxidative gels.

| Effects of IL-10@CMCC on retinal histology after I/R injury
As shown in Figure 5A The immunofluorescence staining of Csp3 revealed the apoptosis in retina ( Figure 5B). The red dots were notably emerged in I/R group, compared with the normal one. However, the number and distribution of red stains were decreased in I/R + Gel group. Moreover, a further decrease was found in the I/R + Gel/IL-10 group.
Furthermore, as shown in Figure 5C, Western blotting of apoptosisrelated proteins clearly showed that the introduction of gels significantly elevated the expression of anti-apoptotic protein, for example Bcl-2 and lowered that of apoptotic proteins, for example Csp3 and Bax. Such modulation was further enhanced by loading IL-10, indicating the protective effect of antioxidative gels and IL-10 against the retinal apoptosis induced by I/R.

| Effects of IL-10@CMCC on the expression of inflammatory cytokine
At day 3, as shown in Figure 6A-E, the mRNA expression levels of TNF-α, IL-1β, iNOS, ICAM-1 and MCP-1 were much higher in I/Rinjured group than the normal rats. In I/R + Gel, the expression of TNF-α, IL-1β, iNOS, ICAM-1 and MCP-1 significantly reduced, compared with that in I/R group (P < .05). Moreover, the mRNA levels of

| Effects of IL-10@CMCC on the level of ROS and the activity of anti-oxidases
The changes in oxidative states in retinas were finally assessed by evaluating the level of ROS and the activity of antioxidant enzymes.
Lucigenin-and luminol-enhanced chemiluminescence (CL) methods revealed the in vivo ROS level at days 3, 7 and 14 respectively. As shown in Figure 7, I/R injury could significantly trigger the CL in retina. The administration of antioxidative gels and IL-10 remarkably decreased the luminol-and lucigenin-enhanced CL signal over 2 weeks, compared with I/R group. Conversely, the activity of SOD and CAT as well as the level of GSH in retina was found significantly reduced in I/R group, compared with those in normal group, as shown in Figure 7C,D. Such significant decrease in the activity of SOD and CAT enzymes and GSH level induced by I/R injury was considerably inhibited by the supplementation with antioxidative gels, CMCC, particularly loaded with IL-10 at days 3, 7 and 14.

| DISCUSSION
Oxidative stress and inflammation were reported to play key roles in retinal damage caused by I/R injury. 31,32 Hence, drugs or treatments capable of attenuating ROS level or/and inflammation in retina are considered as efficacious and main therapies for retinal I/R injury. 31 H 2 O 2 was regarded as one of the most important species in ROS, due to its stability and key status in ROS-related metabolism. 33,34 Thus, in vitro H 2 O 2 tests were gradually accepted as featured tools for the study of ROS or oxidative stress. 35,36 Here, H 2 O 2 was employed to simulate in vitro oxidative stress microenvironment and F I G U R E 2 Representative TUNEL images of rRECs. The rRECs were pre-treated with or without H 2 O 2 and antioxidative gels. The blue was nucleus staining with DAPI and the green was TUNEL staining, representing apoptotic rRECs. Three groups. Bar scale = 100 μm rRECs were selected as model cells due to its physiological function of retinal vascularization, which is essential for structural and functional recovery of the retina. 37 Our study was aimed at the establishment of a desirable drug slow-release system for the retinal treatment of I/R injury. The antioxidative gel carriers, CMCC, were found to significantly decrease intracellular ROS level and thus apoptosis in rRECs. The following analysis of the apoptotic cytokines offered us evidence in molecular level for the CMCC-based protection on rRECs. The accumulation of intracellular ROS could inhibit the Akt-signal pathway, 38 followed by down-regulating survival signals, including Bcl-2 and activating pro-apoptotic factors, such as Bax and Csp3. 39 Additionally, the high concentration of ROS was conducive to triggering the opening of the mitochondria permeability transition gates, thus leading to the release of apoptosis-activating cytokines. 40  The therapeutic efficiency of this antioxidative gel cargo could be further enhanced by loading drugs. IL-10 was selected for its anti-inflammation drug potentials. 43 Previous studies have already utilized IL-10 to mitigate inflammatory response to I/R injury. 44,45 For instance, the up-regulation of IL-10 through pre-conditioning with CpG-oligonucleotides could mitigate the myocardial I/R injury. 46 In this study, we demonstrated that IL-10 load enhanced the protective effect of CMCC on retinal cells against I/R injury. The restoration of retinal morphology and the decrease in apoptosis confirmed the therapeutic effect of IL-10. In addition, the IL-10@CMCC delivery system synergistically mitigated retinal oxidative stress damage and suppressed the expression of inflammatory cytokines (TNF-α, IL-1β, iNOS, ICAM-1 and MCP-1). The pathogenesis of retinal I/R injury was found to up-regulate the expression of multiple inflammatory mediators, including TNF-α, IL-1β, iNOS, ICAM-1 and MCP-1, which was in accordance with previous reports. 3 TNF-α played a pivotal F I G U R E 5 Effects of IL-10@CMCC on retinal histology at 21 d post-I/R injury. A, Representative retinal section images of control, I/ R + PBS, I/R + Gel or I/R + Gel/IL-10 group were attained by H&E staining and the corresponding quantitative total retinal thickness (from the inner limiting membrane to the pigment epithelium). B, Representative immunofluorescence images specified for Csp3 in the 4 groups. C, Western blotting analysis of apoptotic proteins of rRECs, for example Csp3, Bcl-2 and Bax, in the 4 groups. n = 5, bar scale = 50 μm, a P < .05 compared with I/R + PBS group; aa P < .01 compared with I/R + PBS group; b P < .05 compared with I/R + Gel group; bb P < .01 compared with I/R + Gel group